By age 16, close to 2 children in 3 have suffered at least one adverse experience such as parental death, life-threatening illness, or family violence. Adversities have been robustly linked to an array of psychiatric and other medical conditions where the consequences can persist far into adulthood. The medical costs, mental health utilization, societal cost, and the psychological toll on its victims are tremendous. It is not wel understood how early adverse experiences are biologically embedded and what processes might be set into effect that would sustain long term health risks. To address these key questions we need prospective, longitudinal studies that begin in childhood and continue into adulthood and where data on adverse experiences can be linked to biosamples collected before and after adverse experiences as well as in adulthood. We propose just such a study, using already available samples from the Great Smoky Mountains Study (GSMS) and DNA methylation as the biological mechanism of interest. Methylation involves the addition of a methyl group to DNA and, in human non-embryonic cells, occurs mainly at CpGs. Animal and human research have shown that adverse events can result in persistent methylation changes with long-term phenotypic consequences. Capitalizing on these observations we propose a comprehensive study in a real life setting. First, we will use next-generation sequencing (NGS) to assay all >28 million CpGs in the human genome to study adversity-induced methylation changes and their persistence over time. To avoid false positive findings caused by pre-existing case-control differences (e.g. personality related or environmental factors such as poverty) we use a design that considers within-subject changes before and after DSM-IV extreme stressor events. Random assignment to trauma being impossible, this natural experiment is arguably the next best option this topic in children. Consistent with a model assuming a mediator role of methylation, we will select only the methylation sites that changed as a result of adversity for association testing with health risks. For the substantive and methodological reasons, we propose to treat maltreated children as a separate group in these analyses. Finally, we will replicate the 175 top findings in independent samples using a different and targeted technology to minimize the risk of false positives due to sampling and/or possibly technical errors. Successful completion of this project implies that we gained insight into how childhood adversities alters the methylome and what changes persist over time. We will also have identified processes associated with health risks in childhood/adulthood and found replicable methylation biomarkers associated with these risks. Methylation markers are stable and can be measured cost-effectively in blood, which is relatively easy to collect. Our findings therefore als have considerable translational potential as, for example, diagnostic biomarkers of health risk that could guide intervention strategies.